SLIDE 1 Supplementary Materials Efficient killing of murine pluripotent stem cells by natural killer (NK) cells requires activation by cytokines and partly depends on the activating NK receptor NKG2D
Carina Gröschel, Daniela Hübscher, Jessica Nolte, Sebastian Monecke, André Sasse, Leslie Elsner, Walter Paulus, Claudia Trenkwalder, Bojan Polic, Ahmed Mansouri, Kaomei Guan, Ralf Dressel* * Correspondence: Ralf Dressel: rdresse@gwdg.de 1 Supplementary Figures and Tables
- Supplementary Tables 1 to 3
- Supplementary Figures 1 to 6
SLIDE 2 Supplementary Material 2 1.1 Supplementary Tables Supplementary Table 1. Antibodies for immunofluorescence (IF) and immunoblotting (IB) Antigen Isotype Clone Supplier Label Assay Dilution KLF4 rabbit IgG polyclonal (ab34814) Abcam, Cambridge, United Kingdom
1:1000 LIN28 goat IgG polyclonal (YFC01) R&D Systems, Wiesbaden, Germany
1:100 NANOG goat IgG polyclonal (AF2729) R&D Systems, Wiesbaden, Germany
1:200 OCT4 rabbit IgG polyclonal (ab18976) Abcam, Cambridge, United Kingdom
1:1000 SALL4 rabbit IgG polyclonal (ab29112) Abcam, Cambridge, United Kingdom
1:1000 SOX2 rabbit IgG polyclonal (ab59776) Abcam, Cambridge, United Kingdom
1:1000 SSEA1 mouse IgM MC480 Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa, USA
undiluted hybridoma supernatant α- Tubulin mouse IgG1 B-5-1-2 Sigma, Darmstadt, Germany
1:10000 ZFP206 rabbit IgG polyclonal kindly provided by L.W. Stanton, Singapore
1:2000 goat IgG monkey anti-goat IgG polyclonal (705-166- 147) Jackson Laboratories, via Dianova, Hamburg, Germany Cy3 IF 1:600 mouse IgM Goat anti- mouse IgG+IgM polyclonal (115-165- 068) Jackson Laboratories, via Dianova, Hamburg, Germany Cy3 IF 1:600
SLIDE 3
3 mouse IgG Horse anti- mouse IgG polyclonal (#7076) Cell Signaling Technology, Danvers, Massachusetts, USA HRP IB 1:10000
SLIDE 4
Supplementary Material 4 Supplementary Table 2. Primers used for RT-PCR or qPCR Gene Sequence 5'-3' Assay Afp F: CCC ACC CTT CCA GTT TCC RT-PCR R: TAC TGA GCA GCC AAG G Flk1 F: CCT ACC CCA CAC ATT ACA TGG RT-PCR R: TTT TCC TGG GCA CCT TCT ATT Gapdh F: GCA GTG GCA AAG TGG AGA TT RT-PCR R: TCT CCA TGG TGG TGA AGA CA Hprt F: AGC CCC AAA ATG GTT AAG GTT GC qPCR R: TTG CAG ATT CAA CTT GCG CTC AT Klf4 F: TCA GGT ACC CCT CTC TCT TCT TTC qPCR R: CGC TTC ATG TGA GAG AGT TCC T Lin28 F: TCC TCC TGT GTC TCC CAT TC RT-PCR R: AGA GTG AGG CCC TGT CTC AA Mash1 (Ascl1) F: CTC GTC CTC TCC GGA ACT GAT G RT-PCR R: CGA CAG GAC GCC GCG CTG AAA G Myh6 F: CTG CTG GAG AGG TTA TTC CTC G RT-PCR R: GGA AGA GTG AGC GGC GCA TCA AGG Nanog F: AGG GTC TGC TAC TGA GAT GCT CTG RT-PCR R: CAA CCA CTG GTT TTT CTG CCA CCG Nanog F: TTA CAA GGG TCT GCT ACT GAG ATG qPCR R: CAG GAC TTG AGA GCT TTT GTT TG Oct4 F: GGC GTT CTC TTT GGA AAG GTG TTC RT-PCR R: CTC GAA CCA CAT CCT TCT CT Oct4 F: CGG AAG AGA AAG CGA ACT AGC qPCR R: GCC TCA TAC TCT TCT CGT TGG Sox2 F: GGC GGC AAC CAG AAG AAC AG RT-PCR R: GCT TGG CCT CG TCG ATG AAC Zfp206 F: GAG AGG AGG TGG TAC AGC TAT TG qPCR R: AGG TGG AGG TAA CTC ATT CAG TG
SLIDE 5 5 Supplementary Table 3. Antibodies and isotype controls used for flow cytometry Antigen Isotype Clone Label Supplier CD3 rat IgG2b 17A2 FITC BioLegend, Fell, Germany CD49b rat IgM DX5 PE BioLegend, Fell, Germany CD112 rat IgG2a 502-57
- Santa Cruz, Heidelberg, Germany
CD155 rat IgG2a TX56
CD314 (NKG2D) mouse IgG1 149810 PE R&D Systems, Wiesbaden, Germany H2Kb mouse IgG2a AF6-885 PE BioLegend, Fell, Germany H2Db mouse IgG2b KH95 PE BioLegend, Fell, Germany H60 rat IgG2a 205326
- R&D Systems, Wiesbaden, Germany
MULT-1 rat IgG2a 205326 R&D Systems, Wiesbaden, Germany RAE-1 rat IgG2a 186107
- R&D Systems, Wiesbaden, Germany
rat IgG goat IgG polyclonal (112-095-062) FITC Jackson Laboratories, via Dianova, Hamburg, Germany
MOPC-21 PE BioLegend, Fell, Germany
MOPC-173 PE BioLegend, Fell, Germany
MPC-11 PE BioLegend, Fell, Germany
RTK4530 FITC BioLegend, Fell, Germany
RTK2118 PE BioLegend, Fell, Germany The following abbreviations are used: FITC, fluorescein isothiocyanate, and PE, phycoerythrin.
SLIDE 6
Supplementary Material 6 1.2 Supplementary Figures Supplementary Figure 1. The average percentage and SD of CD49b+CD3- NK cells among splenocytes (C57BL/6: n=26 and Klrk1-/-: n=25) and MACS-purified cells (MACS+, n=10) of C57BL/6 and Klrk1-/- mice is shown. Splenocytes of two to three mice were used for one MACS separation.
SLIDE 7
7 A B C Supplementary Figure 2. The iPSC lines used for autologous transplantation are pluripotent. (A) The expression of pluripotency marker genes (Oct4, Sox2, Nanog, and Lin28) and the housekeeping gene Gapdh was determined by RT-PCR in fibroblasts and iPSC clones derived from these fibroblasts. This is exemplified here for the fibroblasts F6 and F8 of two donor mice and in two iPSC clones derived from these fibroblasts (6-4, 6-5 and 8-6, 8-7). (B) The iPSCs (d0) were differentiated in hanging drops and in suspension for 5 days (d5) and subsequently cultured on 0.1% gelatin-coated dishes for further 5, 15, or 25 days (d5+5, d5+15, d5+25). The expression of marker genes for endoderm (Afp), ectoderm (Mash1), and mesoderm (Flk) was analyzed by RT-PCR as illustrated here for the iPSC line 0-3. Expression of alpha-Mhc (Myh6) indicates a differentiation into cardiomyocytes. Gapdh was amplified as housekeeping gene and MEFs served as negative control for the marker genes. (C) Cells of the iPSC lines were subcutaneously injected into immunodeficient RAG2-/- mice and teratomas were obtained after 35 to 91 days. For iPSC line 1-2, the mesodermal differentiation into cartilage (a), endodermal differentiation into intestinal epithelium (b), and ectodermal differentiation into neural rosettes (c) is exemplified here. The scale bar indicates 100 µm.
SLIDE 8 Supplementary Material 8 Supplementary Figure 3. The newly generated ESC line BTL1 expresses pluripotency markers. (A) The expression of pluripotency marker genes (Oct4, Nanog, Klf1, and Zfp206) was determined in parallel to the housekeeping gene Hprt by qPCR in the ESC line BTL1. The mean relative expression
- f two biological replicates is shown compared to the long established ESC line R1. (B) The expression
- f the pluripotency marker proteins OCT4, SALL4, SOX2, KLF4, and ZFP206 in ESC BTL1 cells is
demonstrated by immunoblotting. The expression of α-Tubulin is shown as loading control.
SLIDE 9
9 Supplementary Figure 4. Cells of the stem cell lines iPSC 129Sv, iPSC C57BL/6, and ESC BTL1 were subcutaneously injected into immunodeficient SCID/beige mice and resulting tumors were sectioned and stained with H&E. For each cell line, an ectodermal differentiation (keratinized epithelium), endodermal differentiation (intestinal epithelium), and mesodermal differentiation (cartilage or muscle cells) is shown. The black scale bars indicate 50 µm and the white scale bar 20 µm.
SLIDE 10
Supplementary Material 10 Supplementary Figure 5. A summary of means and SEM of specific lysis of (A) ESCs, (B) iPSCs, and (C) maGSCs by freshly purified NK cells (day 0) or IL-2-activated NK cells (day 4) from C57BL/6 wild type mice is shown as determined by 51Cr-release assays. P-values for the comparisons (2-way- ANOVA adjusted for E:T ratios) are indicated for the comparison of killing by resting and IL-2- activated NK cells.
SLIDE 11
11 Supplementary Figure 6. A summary of means and SEM of specific lysis of (A) ESC BTL1 cells, (B) ESC MPI-II cells, (C) iPSC 129Sv cells, (D) iPSC C57BL/6 cells, (E) maGSC 129Sv cells, and (F) maGSC C57BL/6 cells by freshly purified NK cells (day 0) or IL-2-activated NK cells (day 4) from C57BL/6 wild type (wt) or Klrk1-/- mice (ko) is shown as determined by 51Cr-release assays. P-values (2-way-ANOVA adjusted for E:T ratios) are indicated for the comparison of killing by resting and wild type NK cells (wt day) as well as by resting wild type and NKG2D-deficient NK cells (day 0 killer) and IL-2-activated wild type and NKG2D-deficient NK cells (day 4 killer).
